Atlas of Genetics and Cytogenetics

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Gene Section Review

RPA2 (replication protein A2, 32kDa) Anar KZ Murphy, James A Borowiec Dept of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, New York 10016, USA (AKZM, JAB)

Published in Atlas Database: April 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/RPA2ID42146ch1p35.html DOI: 10.4267/2042/44940 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

The coding sequence is contained within nine exons. Identity There is no confirmed alternative splicing of the RPA2 Other names: REPA2, RPA32 gene, or differential promoter usage. HGNC (Hugo): RPA2 Transcription Location: 1p35.3 The RPA2 mRNA transcript is 1.5 kb. The RPA2 Local order: The human RPA2 gene maps on 1p35.3 promoter contains four E2F consensus sequences between the SMPDL3B (sphingomyelin within the region about 400 bp upstream of the mRNA phosphodiesterase, acid-like 3B) and C1orf38 start site, and putative binding sites for ATF-1 and SP-1 (interferon-gamma inducible gene ICB-1 (induced by transcription factors. Expression is upregulated 2 to 3- contact to basement membrane)). fold by E2F, with mutation of the three start site- proximal E2F sites causing a loss of E2F DNA/RNA responsiveness (Kalma et al., 2001). Description Pseudogene The RPA2 gene is contained within 24.5 kb of RPA2 does not have known pseudogenes. chromosome 1.

The sequence numbering corresponds to EMBL locus DQ001128 (26.6 kb). Exon are indicated as boxes (yellow = 5' UTR, blue = CDS, red = 3' UTR), and introns with orange lines. Two lengthy introns have been truncated (indicated with parallel diagonal lines) to improve viewability.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1) 55 RPA2 (replication protein A2, 32kDa) Murphy AKZ, Borowiec JA

regulate RPA activity in DNA replication and repair Protein reactions, through the RPA2 phosphorylation state (see below). 1) RPA2 phosphorylation. The N-terminal 33 residues of RPA2 contain seven phosphorylation sites. In interphase cells, genotoxic stress (e.g., caused by chromosomal double-strand DNA breaks or DNA replication stress) induces RPA2 phosphorylation by members of the phosphatidylinositol 3-kinase-like kinase (PIKK; ATM, ATR, and DNA-PK) and cyclin- dependent kinases (CDK) families (reviewed in Binz et al., 2004). Mutation of particular RPA2 phosphorylation sites causes defects in homologous recombination (Lee et al., 2010), and Rad51 recruitment to nuclear repair foci (Anantha et al., 2008; Upper panel: Schematic showing the key domains of RPA2. Lee et al., 2010). Mutation of these sites also causes Lower panel: RPA2 phosphorylation sites are shown in bold, genomic instability in response to DNA replication with the primary responsible kinases indicated above each site. stress induced by cellular treatment with hydroxyurea Some sites can be phosphorylated by more than one kinase (e.g., T21 by ATM and DNA-PK). (Vassin et al., 2009). RPA phosphorylation also increases cell viability in response to DNA damage Description arising during mitosis (Anantha et al., 2008). RPA2 is the middle subunit of the heterotrimeric Modification of sites in the phosphorylation region of Replication Protein A (RPA; (reviewed in Binz et al., RPA2 proceeds in a favored order in response to 2004)). The subunit is composed of 270 residues, and genotoxic stress (Anantha et al., 2007). The has a nominal molecular weight of 29.2 kDa. RPA2 phosphorylation of individual RPA2 residues is contains an N-terminal phosphorylation region with 7 dependent on the type of DNA damage or replication phosphorylation sites, a central DNA-binding domain stress encountered (Anantha et al., 2007; Vassin et al., (termed DBD-D), and a C-terminal region that can 2009). RPA2 is a substrate both for PP2A and PP4 form a three-helix bundle. One helix of the three helix phosphatases (Feng et al., 2009; Lee et al., 2010). bundle is contributed by each RPA subunit, with this 2) Involvement of RPA2 in protein-protein structure responsible for supporting heterotrimerization interactions. RPA2 interacts with the nucleotide of the RPA complex (Bochkareva et al., 2002). At least excision repair factor XPA (He et al., 1995), base in the non-phosphorylated state, the N-terminal region excision repair enzyme UNG2 (Mer et al., 2000), is unstructured. DBD-D is constructed from an homologous recombination (HR) factor Rad52 (Mer et oligonucleotide/oligosaccharide binding (OB) fold al., 2000), replication checkpoint protein Tipin (Unsal- (Bochkarev et al., 1999), one of six OB folds found Kacmaz et al., 2007), and the annealing helicase with the RPA heterotrimer (four OB folds are located HARP/SMARCAL1 (Bansbach et al., 2009; Ciccia et in RPA1, and one within RPA3). al., 2009; Yuan et al., 2009). These interactions likely Expression aid the multiple roles of RPA in facilitating DNA repair. RPA is an essential factor for DNA replication and repair, and hence is expressed in all tissues. Homology Localisation A close homolog of RPA2, termed RPA4, is located on Xq21.33 (Haring et al., 2010). Nuclear. Function Mutations General function: RPA is a heterotrimeric single- Note stranded DNA (ssDNA) binding protein that is Naturally-occurring mutations of human RPA2 have essential for chromosomal DNA replication, not yet been described. A small number of genetic homologous recombination, and particular DNA repair polymorphisms have been described in SNP datasets reactions (nucleotide excision repair). The apparent (Y14S, G15R, and N203S), but these have not yet been association constant of the RPA: ssDNA complex is 9 11 -1 reported to have any biological effects (NIEHS SNPs 10 - 10 M (Kim et al., 1992). While RPA2 contains program). a central DBD (Philipova et al., 1996), the major effect of mutating DBD-D is to decrease the size of the Implicated in ssDNA occluded by RPA binding, with only minor effects on RPA: ssDNA affinity (Bastin-Shanower and Colorectal adenocarcinoma Brill, 2001). A key function of the RPA2 subunit is to Disease

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(1) 56 RPA2 (replication protein A2, 32kDa) Murphy AKZ, Borowiec JA

Overexpression of the RPA2 (and RPA1) proteins have Systemic lupus erythematosus (SLE) been found to be prognostic indicator of colon cancer. Disease Strong associations between RPA2 expression and One out of 55 individuals with autoimmune disorders disease stage, lymph node metastasis, and the was found to test positive for anti-RPA2 antibodies histological grade of carcinomas have been observed. (1.8%). This individual had SLE, and secondary Prognosis Sjögren syndrome (Garcia-Lozano et al., 1995). In addition, RPA2 protein expression correlates with poor survival of stage II and III patients (Givalos et al., Rheumatoid arthritis (RA) 2007). Note Ductal breast carcinoma Fibroblast-like synoviocytes (FLSs) are a cell type whose invasive properties provide an indicator of RA Disease severity. Microarray studies from FLSs in DA rats Levels of anti-RPA2 antibodies was observed to be (arthritis-susceptible inbred model) show a modest significantly higher in sera from breast cancer patients increase in the level of RPA2 mRNA, compared to (10.9%; n = 801) as compared to normal controls back-crossed arthritis-resistant DA.F344 (Cia5d) (0.0%; n = 221). Examining individuals with early congenic strains (Laragione et al., 2008). stage intraductal in situ carcinomas, 10.3% (n = 39) similarly showed the presence of high levels of anti- RPA2 antibodies. Even so, follow-up studies indicated References that there were no apparent differences in mean Kim C, Snyder RO, Wold MS. Binding properties of replication survival, occurrences of a second primary tumor, or protein A from human and yeast cells. Mol Cell Biol. 1992 metastasis frequency between breast cancer patients Jul;12(7):3050-9 that were positive or negative for anti-RPA2 sera. Garcia-Lozano R, Gonzalez-Escribano F, Sanchez-Roman J, Although RPA is a nuclear protein, RPA was seen to be Wichmann I, Nuñez-Roldan A. Presence of antibodies to different subunits of replication protein A in autoimmune sera. localized to both nuclei and cytoplasm in the cells of at Proc Natl Acad Sci U S A. 1995 May 23;92(11):5116-20 least one breast tumor, with RPA also over-expressed (Tomkiel et al., 2002). He Z, Henricksen LA, Wold MS, Ingles CJ. RPA involvement in the damage-recognition and incision steps of nucleotide Non-small cell carcinoma excision repair. Nature. 1995 Apr 6;374(6522):566-9 Disease Philipova D, Mullen JR, Maniar HS, Lu J, Gu C, Brill SJ. A hierarchy of SSB protomers in replication protein A. Genes A fraction of individuals with squamous cell lung Dev. 1996 Sep 1;10(17):2222-33 cancer were found to have significant levels of anti- RPA2 antibodies (9.1%; n = 22) (Tomkiel et al., 2002). Bochkarev A, Bochkareva E, Frappier L, Edwards AM. The crystal structure of the complex of replication protein A Laryngeal tumors subunits RPA32 and RPA14 reveals a mechanism for single- stranded DNA binding. EMBO J. 1999 Aug 16;18(16):4498-504 Disease Mer G, Bochkarev A, Gupta R, Bochkareva E, Frappier L, One patient (out of 35; 2.9%) with head and neck Ingles CJ, Edwards AM, Chazin WJ. Structural basis for the tumors tested positive for the presence of anti-RPA2 recognition of DNA repair proteins UNG2, XPA, and RAD52 by sera (Tomkiel et al., 2002). replication factor RPA. Cell. 2000 Oct 27;103(3):449-56 Promyelocytic leukemia Bastin-Shanower SA, Brill SJ. Functional analysis of the four DNA binding domains of replication protein A. The role of Disease RPA2 in ssDNA binding. J Biol Chem. 2001 Sep A derivative of the human HL-60 promyelocytic 28;276(39):36446-53 leukemia cell line (HL-60/P1), selected for its Kalma Y, Marash L, Lamed Y, Ginsberg D. Expression decreased sensitivity to undergo apoptosis in response analysis using DNA microarrays demonstrates that E2F-1 up- to TNF-related apoptosis-inducing ligand (TRAIL), regulates expression of DNA replication genes including was found to have decreased (2-fold) expression of replication protein A2. Oncogene. 2001 Mar 15;20(11):1379-87 RPA2 (Petrak et al., 2009). Bochkareva E, Korolev S, Lees-Miller SP, Bochkarev A. Structure of the RPA trimerization core and its role in the Sjögren syndrome multistep DNA-binding mechanism of RPA. EMBO J. 2002 Apr Disease 2;21(7):1855-63 Serum from a patient with Sjögren syndrome was Tomkiel JE, Alansari H, Tang N, Virgin JB, Yang X, VandeVord found to have high levels of anti-RPA2 antibodies. A P, Karvonen RL, Granda JL, Kraut MJ, Ensley JF, Fernández- Madrid F. Autoimmunity to the M(r) 32,000 subunit of higher rate of non-Hodgkin lymphoma, and lymphoid replication protein A in breast cancer. Clin Cancer Res. 2002 malignancies, is seen in individuals with Sjögren Mar;8(3):752-8 syndrome, compared to normal individuals (Garcia- Lozano et al., 1995).

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Binz SK, Sheehan AM, Wold MS. Replication protein A Feng J, Wakeman T, Yong S, Wu X, Kornbluth S, Wang XF. phosphorylation and the cellular response to DNA damage. Protein phosphatase 2A-dependent dephosphorylation of DNA Repair (Amst). 2004 Aug-Sep;3(8-9):1015-24 replication protein A is required for the repair of DNA breaks induced by replication stress. Mol Cell Biol. 2009 Anantha RW, Vassin VM, Borowiec JA. Sequential and Nov;29(21):5696-709 synergistic modification of human RPA stimulates chromosomal DNA repair. J Biol Chem. 2007 Dec Petrak J, Toman O, Simonova T, Halada P, Cmejla R, Klener 7;282(49):35910-23 P, Zivny J. Identification of molecular targets for selective elimination of TRAIL-resistant leukemia cells. From spots to in Givalos N, Gakiopoulou H, Skliri M, Bousboukea K, vitro assays using TOP15 charts. Proteomics. 2009 Konstantinidou AE, Korkolopoulou P, Lelouda M, Kouraklis G, Nov;9(22):5006-15 Patsouris E, Karatzas G. Replication protein A is an independent prognostic indicator with potential therapeutic Vassin VM, Anantha RW, Sokolova E, Kanner S, Borowiec JA. implications in colon cancer. Mod Pathol. 2007 Feb;20(2):159- Human RPA phosphorylation by ATR stimulates DNA 66 synthesis and prevents ssDNA accumulation during DNA- replication stress. J Cell Sci. 2009 Nov 15;122(Pt 22):4070-80 Unsal-Kaçmaz K, Chastain PD, Qu PP, Minoo P, Cordeiro- Stone M, Sancar A, Kaufmann WK. The human Tim/Tipin Yuan J, Ghosal G, Chen J. The annealing helicase HARP complex coordinates an Intra-S checkpoint response to UV protects stalled replication forks. Genes Dev. 2009 Oct that slows replication fork displacement. Mol Cell Biol. 2007 15;23(20):2394-9 Apr;27(8):3131-42 Haring SJ, Humphreys TD, Wold MS. A naturally occurring Anantha RW, Sokolova E, Borowiec JA. RPA phosphorylation human RPA subunit homolog does not support DNA replication facilitates mitotic exit in response to mitotic DNA damage. Proc or cell-cycle progression. Nucleic Acids Res. 2010 Natl Acad Sci U S A. 2008 Sep 2;105(35):12903-8 Jan;38(3):846-58 Laragione T, Brenner M, Li W, Gulko PS. Cia5d regulates a Lee DH, Pan Y, Kanner S, Sung P, Borowiec JA, Chowdhury new fibroblast-like synoviocyte invasion-associated gene D. A PP4 phosphatase complex dephosphorylates RPA2 to expression signature. Arthritis Res Ther. 2008;10(4):R92 facilitate DNA repair via homologous recombination. Nat Struct Mol Biol. 2010 Mar;17(3):365-72 Bansbach CE, Bétous R, Lovejoy CA, Glick GG, Cortez D. The annealing helicase SMARCAL1 maintains genome integrity at This article should be referenced as such: stalled replication forks. Genes Dev. 2009 Oct 15;23(20):2405- 14 Murphy AKZ, Borowiec JA. RPA2 (replication protein A2, 32kDa). Atlas Genet Cytogenet Oncol Haematol. 2011; Ciccia A, Bredemeyer AL, Sowa ME, Terret ME, Jallepalli PV, 15(1):55-58. Harper JW, Elledge SJ. The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart. Genes Dev. 2009 Oct 15;23(20):2415- 25

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